Embryology - Science topic

Due to the prolifrations and apoptosis ofcells causedby endogenuos factors all organs firstly under go prolifrations and then due to thd effect of this factors organs take tbeir shapes and lumens

Check https://www.sciencedirect.com/science/article/pii/S0166445X21000035

interesting. I did not know. Keep going!

Any antigens present during the period of lymphoid development are exposed to the immune system so the immune system learns to identify and recognise these as "Self", this is the basis of immune tolerance. This includes "chimeric" or more accurately in a genetic context, the antigens of "mosaic" cell populations.

Any antigen which is not introduced at this time will later be identified as foreign - this applies to some of our own antigens that are not recipients of the immune tolerance process, these "sequestered" antigens include those from the lens of the eye and the sperm, so if these are exposed to the immune system later in life, there will be an immune response mounted against them (anti-sperm antibodies are a cause of secondary infertility).

TLDR: If it wasn't there to start with, it will (likely) be rejected.

Hello Riddhi Kirit Pandya,

First of all, what do you call "controls" for RT-qPCR? Housekeeping genes ?

Indeed, results (the relative expression level of each gene) need to be normalized by two housekeeping genes (and not usually only on one); as recommended by the MIQE guidelines (Bustin et al., 2009) with the use of geometric mean to generate an accurate normalization; available at http://www.rdml.org/miqe.php.

Then, be careful to evaluate RT-qPCR results visely (fold-change, relative expression, the 2^(-ΔΔCt), etc...). After computing your RT-qPCR slopes & curves data you obtain your 2^(-ΔΔCt) data. During this procedure, you have to normalise all your data on housekeeping genes (a couple) and on a "control experiment" (associated as the "1:1" value) to compare them as ratio-values: data are presented as relative expression of your genes. --Be careful to use geometric means at this step!--

If you want more more precise and detailed informations, please see Livak and Schmittgen (2001).

Just a thought; evolution is an essential aspect of a dynamic world, i.e., in which time exists. Let us assume two states, A and B occur at different times. From a dynamics point of view, a stable system will reach an equilibrium point after the introduction of certain disruptions in the system. The disruptions cause the system to undergo transient state, i.e., an evolution from one point (A) to another (B) until it reaches an equilibrium point, thereafter the system state becaomes constant if no further disruption is introduced to it. A world can be viewed as a system with a collection of all matters (animated and unanimated) and energy (in all sorts of forms). From time to time certain species of plants, animals or insects emerge while others disappear, however the total mass-energy remains the same in the system. The animals or plants that continue to exist from state to state must evolve according to the criterion (or criteria) imposed by the system, otherwise, they too will completely disappear and replaced by other forms of objects, whose existence fulfill the crietrion set by the system. The question now becomes, in our world what is this criterion? If we know about this criterion, then it is possible to predict our next evelotuion, and the evolution of our enviornments even before they take place.

That certainly depends how you define consciousness. It has been convingly shown that human embryos can perceive pain (which certainly needs some kind of consciousness) at week 20 of prenatal life.

Kind regards, Fres

Dear El-Hassan,

I would suggest to call the deeper end (from where the outgrowth starts) "proximal" and the other end "distal". That should work at least for several organd.

Regards, Fred

Dear Gabor, the following paper may help you:

Mechanical Stresses in Embryonic Tissues: Patterns, Morphogenetic Role, and Involvement in Regulatory Feedback

L.V.Beloussov, S.V.SavelievI, .NaumidiV. V.Novoselov

Hi have a look at the following book:

The Atlas of Chick Development, Third Edition, Ruth Bellairs & Mark Osmond, Academic Press, 2014

I agree with Juan. Have everything at room temperature. I put a drop of Mowiol in the centre of the slide then used a pair of flat tipped angled tweezers to gently lower the coverslip until Mowiol just touches. As the surface tension starts to 'grab' the coverslip let go. Job done.

Hello,

Based on the processes identified in the GO enrichment analyses and the expression of the selected transcripts we suggest that (i) processes related to embryogenic competence and cell wall loosening are involved in activating the cleavage process; (ii) apical-basal polarization is strictly regulated in dominant embryos but not in the subordinate embryos; (iii) the transition from the morphogenic phase to the maturation phase is not completed in subordinate embryos. This is the first genome-wide transcript expression profiling of the earliest stages during embryo development in a Pinus species.

You can read this paper

Elena Candelotti, Paolo De Vito, R. G. Ahmed, Paolo Luly, Paul J. Davis, Jens Z. Pedersen, Hung-Yun Lin, Sandra Incerpi, 2015. Thyroid hormones crosstalk with growth factors: Old facts and new hypotheses. Immun., Endoc. & Metab. Agents in Med. Chem., 15, 71-85.

Thank you for your answer. I also thought so, but recently I've read an article by Johnson et al., 2003 (DOI: 10.1046/j.1525-142X.2003.03048.x), where they wrote that lungfish does not contain germ plasm. But I'm not sure that it is true, since in the same article they wrote that many teleosts including zebrafish lack pelvic appendages, which is definitely a lie.

The quality of the photographs is poor so that  I am uncertain what the photographs are showing. I suppose that you squashed the cells by pressing them. Thus, you do not expect to see the membrane/limits between cells, but some cell elements and also the exploded chromosomic plates. It is recommended the use a phase contrast microscope to observe the chromosomes under the stain that you have used. Please, make sure that you are using the appropriate microscope, the right contrast and the correct section in the squash. It takes a long time, sometimes, to survey the whole preparation, just searching for the correct spots with isolated metaphase chromosomic plates. 

Took me some time, but I solved my problem. I used a mouth pipette and a microinjection needle and applied negative pressure. 

By doing so, I didn't damage the embryo and got rid of the air bubbles.

Ciao Cesare ;-)

yes, it is rather impossible to filter it... I will centrifuge it and fingers crossed!

Thank u very much 4 your suggestion!

Dear Marayam,

Actually, I agree with Audrey Moffett there are many pathways to assess mitochondrial functions. For instance, mitochondrial membrane potential, what I'm doing, is one of the important ways to assess mitochondrial activity.

Best regards,

Usama

In the adult and newborn once the foetal shunts have closed - the stroke volume of the right and left heart are equal and remain so throughout life with only temporary variations that are quickly adjusted in healthy individuals.

This does not mean that the right side (pulmonary) circulation contains an equal volume to the left (systemic) circulation.

Approximately 5L of blood are pumped from the left ventricle each minute in an adult human.  If the equivalent volume was not pumped by the right ventricle each minute then the left heart would have insufficient return to maintain the 5L output per minute. If

left ventricular output decreases transiently while that of the right ventricle remains constant, pulmonary blood volume (and therefore pulmonary pressure) rises until the

outputs of the two ventricles equalise.

The pulmonary circulation always contains less blood than the systemic circulation.

Of approximately 5L blood volume with the heart containing  360mL in diastole (filled, just prior to contraction) the pulmonary circulation holds only 440mL compared to 4200mL in the systemic circulation.  it may help to understand when you realise that the entire right cardiac output goes to  the pulmonary circulation whereas left ventricular output must be divided up amongt the systemic organs: brain; muscle; viscera; etc.This wide distribution contains a lot more blood volume at any one time than the pulmonary circuit but the volume entering and leaving are the same.

Hi. Atefeh

first the most accurate and suitable methods to confirm mice pregnancy is the vaginal plug and if you will not use early embryonic stages from 5-7 days you can confirm from abdomen size 

2- the methods of fixation depends mainly on what kind of experiments you are going to do? please, let me know what kind of experiment so that i can help you

Thanks 

It's a minimum of 100mm/day in the adult rat CNS. Not sure about embryos.

Dear Sarah,

1.       Soluble calcium salts move during the formalin fixation following the concentration gradient.

2.       Insoluble calcium salts may be dissolved in the acid components present in the fixatives, unbuffered formalin fixative has low pH, it contains acids such as formic acid.

3.       The decalcification solves most insoluble calcium salts and depending on the decalcifying solution composition, procedure duration, and washing, some calcium compounds may be redistributed in the object.

Best regards

Pavel

Hi Pooja!

I didn't use mouse embryos before but as far as I know, the protocol of karyotyping (from oocyte till blastocysts) is similar to most of the mammalian embryos.

Try to find this useful book; 

Good luck.

im working with human BM-MSCs not animal 

but check the following links, might help you

For FACS analysis we used treatment with commercially available red blood cell lysis buffer to get rid of enucleated red blood cells. I am not sure if it will work on tissue sections though. 

On day 6 embryo Blastocyst seems to have 84.4+/- 5.7 cells which doubled on day 7 to around 125.5+/-19.

We haven't noticed any difference in outcome, fertilization and implantation.

definitely it has effect on cells and also it matter what quantity of decibels (ultrasound waves can affect the growth of cells)

Hello Sanketh, 

Thank you for responding. This is a great help :) 

Ingrid,

I keep them in containers at room temperature. 

Again, thank you both! :) 

I have seem in an experiment when they exposed seeds to Cobalt 60. When compared with seed killed by heat the membranes shows more integrity.

I would reduce the amplitude and increase the frequency of applications at least by half, e.g use 5V for 20 ms at 250 ms intervals.  You may be irreversibly breaking your membranes so that the cells are dying in large amount and releasing all the cytoplasmic toxins and ATP. I have alot of experience with single cell electroporation and I usually use only about 50 mV to individually electroporate cells (see Kanjhan and Vaney 2008 Pflufers Archive available here at RG). I can fill cells even with voltages as low as 10 mV. Another suggestion is try to reduce or 0 divalent cations, see if it will improve. Let me know how you go:-)

In my opinion, the problem may be in the penetration of the substrate into the whole embryo. Try another method of alkaline phosphatase demonstration.

Best regards.

Pavel

This is quite large number of cases. It is necessary to publish the results. Pavel

Literature on switching genes on and off is easily available. For introductory text and references the following that is available on research gate can be referred to:

Sharma K. Lamarckism Revisited: Epigenetic Interface with Environment Challenges ‘Gene as Fate Theory’ and Rewrites the Rules of Disease, Heredity, and Identity: A Case Study of Discordant Monozygotic Adult Twin Pair for a Psychiatric Disorder. Indian J. Phys. Anthrop. Hum. Genet. 30:167-181.

Platelets are derived from mesoderm. They were used as a proxy measure of 5-HT handling as they take up, store and release 5-HT - they use it for vasoconstriction and clotting. Thus one can look at drugs that target 5-HT handling - SSRIs effect on reuptake, or receptor binding assays etc. - but using platelets as a peripheral measure that is easy to sample repeatedly.

Thank you very much for your interesting answer. I also would like to know what amount of alcohol leads to FAS. Is any scientific data? And whether it is worse to drink at the beginning of pregnancy than later on?Thanks.

Our work with Fetal DNA non invasive isolation at 5 weeks gestation might be of some help.Reference is Trophoblast retrieval and isolation

from the cervix (TRIC) for noninvasive

prenatal screening at 5 to 20 weeks

of gestation

Fertility and Sterility® Vol. 102, No. 1, July 2014 0015-0282/$36.00

We were also using a nylon mesh (Nitex 1000µm) held to a the bottom of a plastic dish by melting mesh extrmities with a hot Pasteur pipette. Now we are just orientating oocytes or embryo with a forceps hold with left hand while injecting with the right hand and a foot pedal.

Bird eggs "Candling" - (ovoscope) will differentiate a non fertilized egg  from fertilized egg.

To find out if the embryo is viable or not - immerse the egg in a deep bowl of water at 100F - if the embryo is viable it will bob up and down. If non viable it will sink.

Sorry this is not molecular/genetic level  but a practical way. Though I don't deal with aviary    

Hello

find links here , i hope help you

Good Luck

We have 3 publications for selecting trophoblasts form maternal cells to examine fetal DNA, Improved fertility in cattle as well as pigs

Henry clemasso@gmail.com

Biomarker-Based Nanotechnology for the Improvement

of Reproductive Performance in Beef and Dairy Cattle

Lectin-Functionalized Magnetic Iron Oxide Nanoparticles for Reproductive

Improvement

Jean M Feugang1*, Shengfa F Liao1, Mark A Crenshaw1, Henry Clemente2, Scott T Willard1,3 and Peter L Ryan1,4

Trophoblast retrieval and isolation

from the cervix (TRIC) for noninvasive

prenatal screening at 5 to 20 weeks

of gestation

Even if my work was mostly devoted to the vascular network of the uterus, you can find more images of these techniques, in the following link:

(Hope this was useful),

Kind regards, 

Maria

Preimplantation development is a specific term and refers to all stages prior to the blastocyst becoming implanted into the uterine wall.  In mouse this would be approximately the first 4-5 days after fertilization.   Early development is not a specific term and is often used to describe events prior to gastrulation, but the use of this term is not very well defined.

Dear Mohammad,

in my oppinion the answer is no. The origin of cord blood is the fetus, therefore there should be no parts of maternal physiology there.

However, in some cases we have observed contamination of cord blood with maternal leucocytes. This might happen in case of peripartal maternofetal transfusion. Those "contaminations" are rare and normally only show up when HLA typing is done, and all of a sudden the child presents with three different haplotypes, two of which are those of the mother...

During the last 11.000 cord blood preparations at our site I remember having observed something like two or three cases.

Hello

Isolate the preimplantation mouse embryos ( 2-, 4- and 8-cell embryos, and blastocysts) in M2 media.

Fixation: Fix embryos in methanol at -20°C for 1h (or overnight).

Wash embryos in 3 drops of M2 media.

Wash embryos in 3 drops of PBS + 0.1% BSA.

Permeabilisation. Incubate embryos for 5min in PBS + 0.1% BSA + 0.1% Triton X-100. (I have not tried but I think this step can be avoided since methanol already permeabilises cells).

Wash embryos in 3 drops of PBS + 0.1% BSA.

Blocking. Incubate embryos in blocking solution (PBS + 0.1% BSA + 10% goat serum) for 1-2h.

1st Primary Antibody. Incubate embryos in the first antibody for 2-4h at RT. In this case we used a mouse anti-Aurora2 (IAK1, DB-transduction lab, cat.no. 610939 - 250 micrograms/ml) diluted 1:100 in blocking solution.

Wash embryos in 3 drops of PBS + 0.1% BSA.

Secondary Antibody. Incubate embryos in the secondary antibody for 2h at RT. In this case we used an anti-mouse-Cy3-conjugated (FluoroLinkTM CyTM3 labelled goat anti-mouse IgG(H+L), Amersham Bioscience cat.no. PA43002) diluted 1:500.

Wash embryos in 3 drops of PBS + 0.1% BSA.

Detection of tubulin. Incubate embryo with anti-tubulin-FITC (SIGMA clone DM1A F-2168) for 2h at RT. Dilution 1:25.

Wash embryos in 4 drops of PBS + 0.1% BSA.

DNA staining. Incubate embryos with DRAQ5 (Alexis) or DAPI (1mg/ml) diluted 1:500 in PBS + 0.1% BSA.

Wash embryos in 2 drops of PBS + 0.1% BSA.

Wash embryos in 2 additional drops of PBS alone.

Mounting. Place embryos on a slide and remove as much PBS as possible. Then add 10-30 microliters of Mowiol and place the coverslip.

NOTE: When embryos are in solutions containing PBS + 0.1% BSA there are no major problems transferring them from drop to drop because they are not sticky. Critical steps in which the embryos can be lost are during fixation, the first washes in M2, and in the last washes in PBS alone before mounting.

Good Luck

“Seed and Soil Theory” is well-known to understand the preferential distant organ/ tissue in which the particular kind of cancer cells forms the metastatic foci. Further, the genetic/epigenetic alteration during the embryonic development is responsible for aggressive cancer development. The tumor involved in skeletal muscle is frequently childhood-onset such as rhabdomyosarcoma etc, but the distant metastasis to the muscle is not common among several kinds of tumors for precisely unknown reason.

From my experience; using feeder cells maintain the normal morphology of oligopotent ESCs (trophoblasts) however using Matrigel (basement membrane matrix) stimulate differentiation into elongated spindle like cells. 

I would like to share a time-lapse video I captured during my experiment.

Load the cells at 37C, it is much more efficient. If you want absolute Levels of calcium, you have to calibrate your experimental setup by chelating all calcium with EGTA (to substract background fluorescence) and known amounts of calcium (make sure to look at the Kd of the indicators). You would first record the fluorescence signal for your cells (your "concentration"), then permealize them with ionomycin and add EGTA (background value) and finally add known concentrations of calcium in extracellular buffer (calibration values). You can then fit the values and determine your absolute intracellular calcium concentration.

Also have a look here:

Good luck!

Dear Gnanasekaran,

The wall of intestine developes from the endoderm-derived epithelium containing specialized intestinal stem cells, mesoderm-derived smooth muscle, vasculature, lymphatics and immune cells and fibrous connective tissue, and the ectoderm-derived enteric nervous system. Therefore also the mutations may be in each cell type and  tumors of the gut can be of the endodermal, mesodermal (mesenchymal) as well as ectodermal origin.

Best regards.

Pavel

depends on what it is you want to look at.  i generally avoid organic stuff unless absolutely necessary.  fixation is typically done before staining and never after.  these are just my own experiences but it really depends.  microtubules are really hard but can be done with organics and FRESH NaBH4.

The Levin lab web page discusses our main interest, which is morphogenesis, the generation of shape during development and regeneration.  And I agree - lacking a standardized way of describing shapes inhibits interdisciplinary collaboration; in fact, it inhibits intradisciplinary comparisons.  Planform  ( http://planform.daniel-lobo.com) is one of the lab's attempts to standardize the description of planarian shape so that experiments can be compared.  In that case, the model is for one person to establish the standard and everyone agrees to participate.  We hope it will work.  

Am I correct that it is descriptions of 3D shape that we are talking about standardizing? There are a lot of issues under discussion here: how to describe shape as well as how to get everyone, in all fields,  to describe shape in the same way.  The former is the more easily addressed, the latter is about human nature, which is way over my head.   Metrology, the study of measurement, is out there.  There are methods for describing shape, geometric morphometrics being the one I know best.  I'm sure the topologists could teach us about shape descriptions.  But I think it has to come from a recognized authority.  NIST and the international body of meteorologists might have some very useful information on deciding on a standard and making everyone stick to it.  Ultimately, though, I believe it will be up to the Natures, Sciences, Cells, and the equivalent players in other fields, to make the editorial decision that they will not accept non-standardized descriptions.  If we must do it to publish, we will do it.  Given that Nature will publish graphs showing the s.e.m. instead of the standard deviation, I think it is an uphill battle.    

A mechanical clock is involved in spine development.

Regards,

Joachim

add insulin, transferrin and selenium (ITS) or supplement with GDF9 and BMP15

Dear Valentin, try to discover the origin of the fragmentation. You might be faced with a sub-optimal culture system. Presence of high glucose concentration is a reason for fragmentation. By removing the fragments you are doing just cosmetic surgery to the embryo but you are not improving it. There is very few evidence that you will have better clinical outcome after fragment removal. www.quartec.co

Hi Daniel,

You have to look for the ovary under the Kidney (or follow the genital tract from the beginning, it will be helpful). You have to use binocular microscopy during the dissection in order to see it

Good luck

Ayhan

The morphology of embryos would be affected by the presence of contamination in the IVC medium, it will slow down the growth of blastomeres and even completely stop the growth of embryos. The contamination can come from different sources such as unsterilized micro-pipettes, contaminated mineral oil, petridishes, CO2 incubator etc. Hence extreme precaution should be taken in maintaining the sterile conditions in the IVF lab.

In the mouse, the PGCs originate from few cells that are localized in the proximal part of the epiblast (an epithelial tissue) that can be detected as early as E6.25 (so at the time of gastrulation). At E7.5, these cells are localized at the basis of the allantois (extraembryonic mesoderm), at the posterior, and are about 50. Contrary to the chick, these cells do not enter the blood flow, but migrate (as single cells but as a collective wave) along the dorsal part of the hindgut until they reach the genital ridges that will form the gonads. During gonad differentiation, germ cells are associated with the granulosa (female) or Sertoli (male) cells, but they always remains as single cells. The only exception is during male spermatogeneis where it is known that germ cells are actually connected to each other via cellular bridges, but not as an epithelium. On the other hand, Sertoli cells do form an epithelium.

Interestingly, there was a recent paper in Nature from Mitinori Saitou's lab that show that PGC-like cells can be induced via the forced expression of some transcription factors and that this pathway does not include a mesodermal step. this would imply that even if in real life, EMT is the first step of PGCs individualization, in vitro, EMT is not absolutely required for this process.

Hi! Look if this papers could help you... Good luck!

Int J Dev Neurosci. 2011 Nov;29(7):723-31. doi: 10.1016/j.ijdevneu.2011.05.012. Epub 2011 Jun 12.

A comparison of proliferative capacity and passaging potential between neural stem and progenitor cellsin adherent and neurosphere cultures.

Sun T, Wang XJ, Xie SS, Zhang DL, Wang XP, Li BQ, Ma W, Xin H.

Stem Cells. 2008 Nov;26(11):2938-44. doi: 10.1634/stemcells.2008-0558. Epub 2008 Aug 28.

Don't look: growing clonal versus nonclonal neural stem cell colonies.

Coles-Takabe BL, Brain I, Purpura KA, Karpowicz P, Zandstra PW, Morshead CM, van der Kooy D.

Sure. Happy to help. This is the citation:

RIGGS HE, RUPP C. Variation in Form of Circle of Willis: The Relation of the Variations to Collateral Circulation: Anatomic Analysis. Arch Neurol. 1963;8(1):8. doi:10.1001/archneur.1963.00460010024002.

We usually have a collection of cross-sections. If your main aim is to showcase the mesentery perhaps suspending the small intestine above the abdominal cavity, with a series of loops around the intestine with fishing line, then tied off on a grate above the cadaver. That way you create space to photograph the mesentery as well as retro and intra peritoneal with rectum, sigmoind, transverse colon etc. That's one way to use an already dissected cadaver with the normal dissection protocol.

A %age well diluted should not harm the embryo. If uve collected much embryos, a try will not harm u... :-)

Discovery of therapeutic targets by phenotype-based zebrafish screens

Randall T. Peterson., Drug Discovery Today: Technologies, 2004.

Peterson et al., 2000. Small molecule developmental screens reveal the logic and timing of vertebrate development. PNAS.

It could be helpful for you to understand about Zebrafish embryos as a disease model to screen novel small molecules

In birds, females lay down medullary bone as the reproduction season approaches, it is then used during the egg-laying period. So, it is typically only present seasonally. I think the case for simply the presence of medullary bone in non-avian dinosaurs is still open for debate.

I hope, that you can find your answer in here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1767747/

In the healthy state, fetomaternal hemorrhage occurs at the time of deliver, not before. The routine fetal hemoglobin stain (Kleihauer Betke) is quite sensitive to the relationship between the amount of cells on the smear and the alkali denaturation process. False negative results can occur when there is too strong an alkali, too long a time of exposure to the solution, or too thin a blood smear. During an uncomplicated delivery, only 30cc or so of fetal blood will enter the maternal circulation and be diluted throughout the circulation so it is possible that the sample used does not contain any fetal cells.

During human gestation, hematopoiesis occurs in the yolk sac (first trimester), the spleen and liver (end of first - all of the second and partially during the third trimester) and in the bone marrow (end of third trimester). During gestation and particularly in the second trimester, it is not uncommon to see immature blood cells in the fetal circulation. It is even possible to see them in the neonate.